Human can form vitamin D3 (cholecalciferol) in the skin with the help of sunlight. Vitamin D2 (ergocalciferol) is taken up with food. Even though vitamin D2 and D3 slightly differ in their side chains they have identical biological activity. They are both bound in circulation by the vitamin D binding protein (VDBP) and metabolised in liver to 25-hydroxyvitamin D. 25-hydroxyvitamin D is the storage form in the body and the vitamin D metabolite with the highest concentration in serum or plasma. When needed, it is hydroxylated in the kidney to 1α,25-dihydroxyvitamin D (the so-called D-hormone) which is the biologically active form and regulating the absorption of calcium in the intestines, the mineralisation of the bones, the differentiation of osteoplasts, the synthesis of bone matrix and, among others, also the neuromuscular functions. Even a tiny deficiency of less than 15 ng 25-hydroxyvitamin D per mL serum (37.5 nmol/L 25-OH-Vit.D/L) causes a rise of the parathormon level and increased bone resorption, due to the reduced calcium absorption (Chapuy M C et al. in J Clin Endocrinol Metab 1996; 81:1129-33). Vitamin D deficiency is an important risk factor for senile osteoporosis. An early diagnosis and vitamin D2 supplementation allow an effective prevention of bone fractures. A heavy vitamin D deficiency of less than 5 ng 25-hydroxyvitamin D per mL serum (12.5 nmol/L) causes rickets in children and osteomalacia in adults (Scharla et al. Exp Clin Endocrinol. Diabetes, 1996, 104:289-292). Excess of vitamin D due to overdosing causes hypercalcaemia. During winter about one third of the population in Germany above 50 years of age suffer from vitamin D deficiency due to the darkness (Scharla et al., Osteoporose Int. 1998; 8 (Supplement 2):S7-S12). Younger people can also suffer from vitamin D deficiency, due to gastro-intestinal diseases, liver dysfunction, maladsorption, or drug-induced heightened metabolism, for example caused by antiepileptica.
Conventional lab techniques for the determination of 25-hydroxyvitamin D in serum and plasma are very laborious (Tanner et al. (1988), J. Assoc. of Analyt. Chem., 17, 607-710). Furthermore, U.S. Pat. No. 5,981,779 (Holick et al), WO 89/01631 and EP 0 583 945 (DeLuca et al) teach vitamin D testing based on the binding to VDBP. To achieve that, the vitamin D metabolites first have to be extracted from the plasma or serum using organic solvents, and then purified by chromatography. WO 99/67211 (Armbruster et al) teaches a sample preparation involving the precipitation of the plasma and serum proteins by ethanol. The protein precipitate is then removed by centrifugation and the ethanol supernatant, comprising the soluble vitamin D metabolites, is used in the binding assay. EP 0 753 743 (Hollis) teaches the preparation of the plasma or serum sample using periodate precipitation. The quantification of the vitamin D compounds is then carried out in the protein-free supernatant. DE 10144 905 (Armbruster et al) describes a method for determining vitamin D directly in plasma or serum wherein a soluble salicylic compound is added to the serum or plasma to release the 25-hydroxyvitamin D from the VDBP. The amount of 25-hydroxyvitamin D in the plasma or serum is then determined directly using antibodies.
The sample preparation methods mentioned above are either laborious or prone to errors or both. It is the object of the present invention to provide a simple and reliable method for the direct quantitative determination of 25-hydroxyvitamin D in serum or plasma.